RU2376368C2 - STRAIN OF BACTERIA Escherichia coli JM109/pHINS21 - PRODUCER OF HYBRID PROTEIN WITH HUMAN PROINSULIN AND METHOD FOR PRODUCTION OF HUMAN PROINSULIN - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: recombinant strain Escherichia coli is produced, which contains plasmid pHINS21 (Escherichia coli JM109/ pHINS21), defining synthesis of hybrid protein, made of N-terminal fragment of human gamma-interferon and human proinsulin, joined by peptide linker, which contains site of splitting with enterokinase (Asp₄Lys). Yield of hybrid product that includes proinsulin, provided with new strain-producer, makes at least 30% of total amount of cell protein. Method is suggested for preparation of human proinsulin, including cultivation of strain-producer Escherichia coli JM109/pHINS21, separation of inclusion bodies and their dissolution, renaturation of hybrid protein and its cleaning with ion-exchange chromatography, splitting of hybrid protein with enterokinase or its catalytic subunit and cleaning of proinsulin by ion-exchange chromatography on sorbates with sulfprofile groups.

EFFECT: invention simplifies technological process for production of recombinant human proinsulin and improves conditions of its execution from the point of view of safety engineering.

4 cl, 4 dwg, 5 ex

Description

The invention relates to the field of biotechnology, namely to the production of recombinant human proinsulin, and can be used in medicine.

Proinsulin is synthesized in the rough endoplasmic reticulum of b-cells of pancreatic islets of Langerhans in the form of a precursor, preproinsulin (molecular weight 11500 Da). The leader sequence, consisting of 23 amino acid residues, directs the precursor molecule to the Golgi apparatus and is cleaved there. As a result, a proinsulin molecule is formed (molecular weight about 9000 Da), which takes the conformation necessary for the proper formation of disulfide bridges. Then, proinsulin is cleaved in several specific sites with the formation of mature insulin and C-peptide, which are deposited in secretory granules.

Recent medical studies have shown that proinsulin can block the destruction of pancreatic b-cells, acting as a local protector, and also stimulate their regeneration. Therefore, the development of methods for producing recombinant human proinsulin for the prevention and treatment of diabetes is extremely relevant.

A known method of producing recombinant human proinsulin [1-3], which includes the lysis of E. coli cells expressing hybrid proteins with human proinsulin in the form of inclusion bodies, dissolution of inclusion bodies in a buffer solution containing urea or guanidine chloride, sulfonation of the hybrid proteins with sodium sulfonate and sodium tetrathionate, centrifugation of the proinsulin S-sulfonate fusion protein, cleavage of the proinsulin S-sulfonate from the leader peptide by treatment with cyanogen bromide, purification of the proinsulin S-sulfonate on-exchange chromatography, re-folding of the proinsulin S-sulfonate chain and purification of human proinsulin by adsorption chromatography.

The disadvantages of the described method are the multi-stage process and the use of a highly toxic reagent - cyanide bromide when cleaving a hybrid protein.

Therefore, there is currently a need to develop improved methods for producing recombinant human proinsulin that are highly productive and non-toxic.

To obtain recombinant proteins from hybrid polypeptides, approaches are known in which the cleavage of the final product is carried out using highly specific proteolytic enzymes. Such methods include the construction of hybrid proteins containing a specific proteinase recognition site between the carrier protein (leader peptide) and the target protein, purification of the synthesized hybrid protein and its enzymatic cleavage. One of the highly specific proteinases used for this purpose is enterokinase (enteropeptidase, EC 3.4.21.9), which cleaves the peptide bond after the (Asp) ₄ Lys sequence.

Description of the invention

The present invention relates to a human proinsulin precursor fusion protein with the sequence shown in FIG. 2, comprising the amino acid sequences of a leader peptide representing the N-terminal fragment of human gamma interferon (SEQ ID NO: 1), a peptide linker (SEQ ID NO: 2) containing a cleavage site of human enterokinase and proinsulin (SEQ ID NO: 3).

In addition, the invention relates to DNA encoding the specified fusion protein, with the nucleotide sequence shown in figure 2.

The invention relates to a recombinant plasmid containing the specified DNA encoding the specified hybrid protein with human proinsulin, as well as including regulatory elements. The following non-limiting elements and markers can be cited as regulatory elements and genetic markers that are used in the plasmid of the present invention: transcription promoter tac, E. coli ribosomal operon transcription terminator, β-lactamase gene (bla) used as a genetic marker , which determines the resistance of ampicillin to bacterial cells transformed by pHINS21 plasmid.

In a particular case, the invention relates to a recombinant plasmid pHINS21 (figure 1) containing the specified DNA encoding a hybrid protein (figure 2), in which the amino acid sequences of the leader peptide, which is an N-terminal fragment of human gamma-interferon, and human proinsulin connected by a peptide linker containing an enterokinase cleavage site, wherein the plasmid has a molecular weight of 2.36 MDa and includes unique recognition sites of restriction endonucleases located at the next distance to the right t EcoRI site: BamHI - 159 bp, HpaI - 185 bp, HindIII - 442 bp, PvuI - 1382 bp

The new hybrid protein provides a high yield of the target product, human proinsulin, due to efficient renaturation and efficient cleavage of the indicated hybrid protein by enterokinase.

The invention also relates to transformed Escherichia coli cells containing said recombinant plasmid, a producer of human proinsulin fusion protein. In a particular case, the invention relates to a strain of bacteria Escherichia coli JM109 / pHINS21 - a producer of a hybrid protein of human proinsulin.

In addition, the claimed fusion protein with human proinsulin, for example, produced by E. coli strain JM109 / pHINS21, can be used in a method for producing human insulin. In this case, a new hybrid protein is transformed into insulin by enzymatic cleavage of the specified hybrid protein by enterokinase, trypsin and carboxypeptidase B.

The invention relates to a new method for producing human proinsulin. The inventive method for producing human proinsulin includes culturing a producer strain of Escherichia coli, destroying bacterial cells, isolating inclusion bodies, dissolving them in a buffer containing urea and dithiothreitol, renaturizing and purifying the renatured fusion protein with human proinsulin, cleaving it with a specific proteinase, followed by purification with subsequent proteinase synthesis, followed by target product. In a particular case, the method involves the cultivation of a new strain of E. coli JM109 / pHINS21 carrying the new plasmid pHINS21, cleavage of the hybrid protein is carried out by enterokinase (or its catalytic subunit), and the proinsulin obtained after enzymatic cleavage is purified by ion-exchange chromatography on SP-Sepharose followed by high-performance high-performance liquid chromatography reverse phase chromatography.

An advantage of the claimed method for producing human proinsulin is the simplification of the process and the exclusion of highly toxic reagent from the technology.

DETAILED DESCRIPTION OF THE INVENTION

A new recombinant plasmid, pHINS21, was created, which determines the synthesis of a hybrid protein with a molecular weight of about 15.85 kDa, in which the amino acid sequences of the leader peptide, which is the N-terminal fragment of human gamma interferon, and human proinsulin are connected by a peptide linker containing the enterokinase cleavage site . A new hybrid protein, the properties of which determine its effective renaturation and efficient enzymatic cleavage by enterokinase, provides a high yield of the target product, human proinsulin.

The optimal linker length and its amino acid composition were determined experimentally.

The recombinant plasmid pHINS21 was constructed on the basis of the known plasmid pHINS05 [4]. The DNA plasmid pHINS05 was subjected to complete hydrolysis with restriction enzymes BclI and HpaI, and the obtained BclI-HpaI fragment (4.2 kb) was ligated with an oligonucleotide duplex obtained by annealing synthetic oligonucleotides: the meaning presented in the sequence SEQ ID NO: 4, and the antisense shown in the sequence of SEQ ID NO: 5. As a result, a nucleotide sequence encoding an enterokinase cleavage site, namely, a peptide fragment (Asp) ₄ Lys, is inserted into the hybrid protein gene in front of the human proinsulin DNA.

The ligation mixture was used to transform competent E. coli JM109 cells. The result was a recombinant plasmid pHINS20, the structure of which was confirmed by restriction analysis. The structure of the gene (FIG. 2) encoding the fusion protein was confirmed by sequencing.

To increase the number of copies of the obtained plasmid pHINS20 in the bacterial cell, it was deleted by the rop gene (negative copy number regulator) [5]. To this end, pHINS20 plasmid DNA was subjected to complete hydrolysis with restriction enzymes Eco47III and SnaI, and the resulting Eco47III-SnaI fragment (3.6 kb) was ligated. The result was a recombinant plasmid pHINS21, the structure of which was confirmed by restriction analysis and sequencing.

The new recombinant plasmid pHINS21 encodes a 143 amino acid fusion protein with a molecular weight of 15.85 kDa, in which the amino acid sequences of the leader peptide, which is the N-terminal fragment of human gamma interferon (SEQ ID NO: 1), and human proinsulin (SEQ ID NO : 3) linked by a peptide linker (SEQ ID NO: 2) containing the enterokinase cleavage site.

The specified plasmid consists of a fragment of BamHI-EcoRI plasmid pKK223-3 [6] containing the tac transcription promoter; an EcoRI-HindIII fragment comprising a fusion protein gene with the nucleotide sequence shown in FIG. 2, encoding the N-terminal fragment of human gamma interferon, a peptide linker with an enterokinase cleavage site and the amino acid sequence of human proinsulin; the HindIII-SnaI fragment of plasmid pKK223-3 containing the transcription terminator of the E. coli ribosomal operon, β-lactamase gene (bla), replication initiation site (ori); and Eco47III-EheI fragment of plasmid pKK223-3; unique recognition sites by restriction endonucleases located at the following distance to the right of the EcoRI site: BamHI - 159 bp, HpaI - 185 bp, HindIII - 442 bp, PvuI - 1382 bp

The strain producing E. coli JM109 / pHINS21 is obtained by transforming E. coli cells of strain JM109 with plasmid pHINS21. After transformation, colonies grown on ampicillin medium are selected, plasmids are isolated from them and subjected to restriction analysis and sequencing. The cell line carrying the plasmid pHINS21 is plated several times on agarose medium supplemented with ampicillin and the resulting monoclonal culture is inoculated with 5 ml of liquid medium with ampicillin. The culture is checked for the presence of inducible expression of the fusion protein, packaged, glycerol is added and stored at minus 40 ° C.

The new strain of Escherichia coli JM109 / pHINS21, carrying the plasmid pHINS21, is a producer of a hybrid protein containing the amino acid sequence of human proinsulin, and is characterized by the following features.

Morphological signs: small, rod-shaped cells, gram-negative, non-spore-bearing, 1 × 3.5 µm in size, motile, with well-defined inclusion bodies after induction of fusion protein synthesis.

Cultural traits: when growing on LB agar medium, the colonies are round, smooth, translucent, shiny, gray. The edge is even, the diameter of the colonies is 1-3 mm, the consistency is pasty. Growth in liquid media (LB, minimal medium with glucose) is characterized by even turbidity.

Physiological and biochemical characteristics: cells grow at a temperature of 4-42 ° C, optimum pH of 6.8-7.6. As a source of nitrogen, both ammonium mineral salts and organic compounds are used: amino acids, peptone, tryptone, yeast extract. When growing on a minimal medium, glycerin, carbohydrates, amino acids are used as a carbon source.

Antibiotic resistance: cells of the producer strain show resistance to ampicillin (up to 500 mg / ml) due to the presence of the β-lactamase (bla) gene in the plasmid.

Stability of the plasmid in the strain. When maintaining the cells for several months on an agarized LB medium containing ampicillin, no plasmid loss or rearrangement affecting the expression of the fusion protein is observed.

In the new strain, the hybrid protein after induced expression accumulates in the form of inclusion bodies, and its content is at least 30% of the total protein of the cell.

The resulting producer strain E. coli JM109 / pHINS21 was deposited in the collection of microorganisms of National Biotechnologies OJSC under number 21-06.

The new recombinant plasmid pHINS21 provides efficient biosynthesis of a hybrid protein containing human proinsulin in other bacterial strains of Escherichia coli , for example, the E. coli BL21 host strain, as described below.

As a non-limiting example, the description provides a method for producing human proinsulin using E. coli JM109 / pHINS21. The cultivation conditions of the producer strain and the isolation of inclusion bodies are not determining, and the specialist in this field, based on general knowledge, will select similar conditions that do not affect the achievement of the technical result.

Cultivation of the culture of the producer strain E. coli JM109 / pHINS21 is carried out on a nutrient medium based on casein hydrolyzate and yeast extract Saccharomyces cerevisiae . To induce the synthesis of a hybrid protein, 1-isopropyl-β-D-1-thiogalactopyranoside (IPTG) is introduced in the middle of the logarithmic growth phase. Cultivation is continued until the desired level of intracellular inclusions of the fusion protein (inclusion body) is formed. After growing, the culture is besieged, the cells are destroyed by one of the acceptable methods, and inclusion bodies are separated by centrifugation. The sediment of inclusion bodies (paste) contains about 5-7% of the proinsulin fusion protein.

Isolation of the hybrid protein from inclusion bodies, its enzymatic cleavage and purification of proinsulin is carried out according to the following scheme:

• inclusion bodies are dissolved in 0.1 M Tris-HCl buffer, pH 8.0, containing 8 M urea, followed by the addition of dithiothreitol to a final concentration of 10 mM;

• the hybrid protein is renatured in a 10-fold volume of 0.1 M glycine buffer, pH 9-11 at a temperature of 10-14 ° C for 20-24 hours;

• purification of the renatured hybrid protein is carried out by chromatography on DEAE-Sepharose FF, equilibrated with 0.05 M Tris-HCl buffer, pH 7.0-7.5, containing 0.15 M NaCl. Protein is eluted with a linear gradient of 0.15-0.4 M sodium chloride in equilibration buffer;

• purified hybrid protein is cleaved with enterokinase (or its catalytic subunit) for 12 to 24 hours at a pH of 7.0-7.5 and a temperature of 16-26 ° C. The reaction is stopped by acidifying the hydrolyzate with hydrochloric acid to a pH of 4.6-5.0, and the resulting precipitate of impurity proteins is separated by centrifugation;

• proinsulin is purified by ion exchange chromatography on SP-Sepharose FF in 0.05-0.2 M ammonium acetate buffer, pH 3.0-6.0, containing 1-2 M urea. The sorbed protein is eluted with a linear gradient of potassium chloride from 0 to 0.5 M in equilibration buffer;

• further purification of proinsulin is carried out by the method of reverse phase high performance liquid chromatography (RP HPLC).

Brief Description of the Figures

Figure 1 presents the physical map of the recombinant plasmid pHINS21.

Figure 2 presents the nucleotide sequence of the gene of the hybrid protein with human proline in the plasmid pHINS21 and its encoded amino acid sequence.

Figure 3 shows the HPLC analysis of the material obtained after purification of the renatured hybrid protein with human proinsulin on DEAE-Sepharose.

Figure 4 shows the HPLC analysis of the cleavage products of the hybrid protein with human proinsulin enterokinase.

Examples

The invention is illustrated by the following non-limiting examples.

Example 1 Construction of the plasmid pHINS21

The plasmid pHINS21 is constructed on the basis of the known plasmid pHINS05 [4]. The pHINS05 plasmid DNA was subjected to complete hydrolysis with restriction enzymes BclI and HpaI. For this, 5 μg of plasmid DNA rHINS05 in 20 μl of a buffer containing 33 mM Tris-acetate, pH 7.9, 66 mM K-acetate, 10 mM Mg-acetate and 0.1 mg / ml BSA (buffer 1) and 10 IU restriction enzymes BclI and HpaI are incubated for 1.0 hour at 37 ° C. A DNA fragment of BclI-HpaI about 4.2 kb in size was isolated from the resulting hydrolyzate. by electrophoresis in a 0.8% gel fusible agarose. Next, the DNA is deproteinized with phenol, a mixture of phenol with chloroform (1: 1), chloroform, precipitated with ethyl alcohol, and dissolved in 20 μl of water. The obtained BclI-HpaI fragment containing the fusion protein gene and the vector part of the plasmid pHINS05 is ligated with a 20-fold molar excess of the oligonucleotide duplex obtained by annealing the synthetic oligonucleotides: sense, shown in the sequence SEQ ID NO: 4, and antisense, shown in the sequence SEQ ID NO: 5.

As a result, a nucleotide sequence encoding an enterokinase cleavage site is inserted in front of the human proinsulin DNA into the hybrid protein gene.

Competent cells of E. coli strain JM109 are transformed with a ligation mixture (10 μl) and plated on LB agar containing 100 μg / ml ampicillin. Plasmid DNA was isolated from grown colonies and sequenced between EcoRI and HpaI restriction enzyme sites to confirm insertion of the fusion protein gene into the linker. The result is the plasmid pHINS20.

To obtain a rop gene deletion plasmid (negative copy regulator), 10 U restriction enzymes Eco47III and SnaI, generating blunt ends, are added to 5 μg of pHINS20 plasmid DNA in 20 μl and the mixture is incubated for 1 hour at 37 ° C. Next, the DNA is deproteinized with phenol, a mixture of phenol with chloroform (1: 1), chloroform, precipitated with ethyl alcohol, and dissolved in 20 μl of water. The DNA thus obtained was ligated in 30 μl of ligation buffer in the presence of 5 U of T4 phage ligase DNA for 16 hours at 8 ° C. The competent cells of the E. coli strain JM109 are transformed with the ligation mixture (10 μl) and plated on LB agar containing 100 μg / ml ampicillin. Bacterial clones carrying plasmid DNA of 3.6 kbp were selected. The isolated plasmids are subjected to restriction analysis and sequenced according to the Sanger method. The result is the plasmid pHINS21.

Example 2 Obtaining strain E. coli JM109 / pHINS21 and determine the level of its productivity

The plasmid pHINS21 transform the competent cells of the E. coli strain JM109 and plated on LB agar containing 100 μg / ml ampicillin. Separately, a localized colony is reseed three times on plates with LB agar containing 100 μg / ml ampicillin. The resulting monoclonal culture was inoculated with 5 ml of liquid LB medium with ampicillin and incubated overnight with vigorous shaking at 37 ° C.

The resulting producer strain E. coli JM109 / pHINS21 was stored in 20% glycerol at minus 40 ° C.

To determine the level of inducible expression of the hybrid protein, the night culture was seeded at a dilution of 1:50 in 5 ml of LB liquid medium containing 100 μg / ml ampicillin and grown to a turbidity of 0.8 at 37 ° C on a rocking chair at 200 rpm. IPTG was added to the culture to a concentration of 1.0 mM and incubation was continued under the same conditions for 3 hours. Cells are collected by centrifugation, the pellet is suspended in a buffer containing 62.5 mM Tris-HCl, pH 6.8, 3% sodium dodecyl sulfate, 5% 2-mercaptoethanol, 10% glycerol and 0.01% bromophenol blue and heated for 3 minutes at boiling water bath. The obtained cell lysate was analyzed by electrophoresis in an 18% polyacrylamide gel with sodium dodecyl sulfate [7]. The gel is stained with Coomassie R-250, scanned and densitometric. According to densitometry, the content of the hybrid protein is at least 30% of the total protein of the cell.

Example 3 Obtaining strain E. coli BL21 / pHINS21 and determine the level of its productivity

Competent cells of the E. coli strain BL21 transform the pHINS21 plasmid DNA and plated on LB agar containing 100 μg / ml ampicillin. Separately, a localized colony is reseed three times on plates with LB agar containing 100 μg / ml ampicillin. The resulting monoclonal culture was inoculated with 5 ml of LB liquid medium and incubated overnight with vigorous shaking at 37 ° C.

The resulting producer strain E. coli BL21 / pHINS21 is stored in 20% glycerol at minus 40 ° C.

The determination of the productivity of the E. coli strain BL21 / pHINS21 is carried out by the same method as for the E. coli strain JM109 / pHINS21 described in Example 2. According to densitometry, the content of the hybrid protein in the induced cells of the obtained strain is at least 30% of the total cell protein .

Example 4 Obtaining human proinsulin when using the producer strain E. coli JM109 / pHINS21

Step 1. Isolation of inclusion bodies containing fusion protein with human proinsulin

A culture of E. coli strain JM109 / pHINS21 is grown on a nutrient medium based on casein hydrolyzate and baker's yeast extract. Induction of the biosynthesis of the hybrid protein is carried out by introducing an IPTG inducer in the middle of the logarithmic phase of the culture growth. Cultivation is continued until the formation of intracellular inclusions of the fusion protein. After cultivation is complete, the culture is precipitated and used to isolate inclusion bodies containing the proinsulin fusion protein.

Cells are suspended in buffer A containing 50 mM sodium disubstituted phosphate, 1 mM EDTA, 0.2 M sodium chloride per 1 g of biomass 5-10 ml of buffer and destroyed on the Gaulin disintegrator. Destroyed cells are centrifuged for 20 min on a Beckman J-30I at 8000 rpm. The resulting pellet was resuspended in buffer A containing 1% Triton X-100, incubated for 1 hour, and the suspension was centrifuged for 20 minutes on a Beckman J-30I at 8000 rpm. The pellet was resuspended in buffer A and pelleted in a centrifuge. The washed precipitate of inclusion bodies (paste) containing about 5% of the proinsulin fusion protein is Packed, frozen and stored at minus 40 ° C.

Step 2. Renaturation and Isolation of the Hybrid Protein

5 g of defrosted inclusion Taurus paste is dissolved in 100 ml of a buffer solution containing 0.1 M Tris-HCl, pH 8.0, 10 mM dithiothreitol and 8 M urea, and incubated with stirring for 18-24 hours at 14 ° C. The solution was centrifuged for 20 min on a Beckman J-30I at a speed of 8000 rpm at 14 ° C. Renaturation of the fusion protein contained in the supernatant is carried out by 10-fold dilution in 0.1 M glycine buffer, pH 9-11. The hybrid protein solution is incubated for 20-24 hours, stirring and maintaining a temperature of 10-14 ° C. The process of renaturation of the hybrid protein is controlled by RP HPLC. After the formation of correctly closed S-S bonds in 50-70% of the molecules of the hybrid protein, the solution is acidified with 2 N. hydrochloric acid to a pH of 7.0-7.2, and the precipitate formed is separated by centrifugation.

The content of the renatured hybrid protein in the supernatant is 112 mg.

The hybrid protein solution is applied to a 100 ml column filled with DEAE-Sepharose FF, pre-equilibrated with 0.05 M Tris-Hcl buffer, pH 7.0-7.5. The column is washed with equilibration buffer containing 0.15 M NaCl. Sorbed protein is eluted with a linear gradient of 0.15-0.4 M sodium chloride in equilibration buffer. Fractions containing the fusion protein are analyzed by RP-HPLC (Figure 3). The result is 95 mg of purified fusion protein.

The correspondence of the obtained hybrid protein with human proinsulin was confirmed by mass spectrometry in coincidence with the calculated molecular weight of the specified protein.

Stage 3. Enzymatic cleavage of the hybrid protein

The enzymatic hydrolysis reaction is carried out at a temperature of 25 ° C with constant stirring. CaCl _{2 was} added to a solution of purified fusion protein to a concentration of 1 mM. Then, a solution of recombinant enterokinase (catalytic subunit of bull enterokinase) (Novagen) is added to the reaction mixture at the rate of 1 IU per 4 mg of protein and incubated for 20 hours. The hydrolysis products of the hybrid protein are analyzed by RP-HPLC. Figure 4 presents the HPLC analysis of the cleavage products of the hybrid protein by enterokinase after 20 hours of incubation. The cleavage reaction is stopped by acidifying the material 2 N. hydrochloric acid to a pH of 4.0-5.0. The resulting precipitate of impurity proteins is separated by centrifugation.

Step 4. Purification of Human Proinsulin

The clarified solution containing proinsulin is applied to a 20 ml chromatographic column filled with SP-Sepharose FF, pre-equilibrated with 0.03 M ammonium acetate buffer, pH 5.0, with 2 M urea. The column is washed with a balancing buffer until the baseline of the control flow densitometer is reached. The adsorbed proinsulin is eluted by a linear gradient of potassium chloride from 0 to 0.5 M in a balancing buffer. The content and purity of human proinsulin in the collected fractions is determined by RP HPLC. The combined fractions contain 54 mg of human proinsulin with a purity of at least 85%.

Further proinsulin purification is carried out by RP HPLC method on a preparative chromatograph ”Armen” (France). A 200 ml column filled with Cromasil C18 is equilibrated with 20% isopropanol with 0.1% trifluoroacetic acid and a 54 mg protein solution of proinsulin from the previous purification step is supplied. The protein is eluted with a gradient of isopropanol (with 0.1% trifluoroacetic acid) from 20 to 40%. The collected fractions of the main peak of proinsulin are analyzed for impurities by RP-HPLC.

After purification, 46 mg of highly purified human proinsulin with a basic substance content of 98% are obtained.

The authenticity of the obtained target product is confirmed by the coincidence of the calculated molecular weight of human proinsulin with the molecular weight of the target product determined by mass spectrometry.

Example 5

Obtained in example 4 (step 2), the hybrid protein with human proinsulin was digested with enterokinase from a natural source.

The enzymatic cleavage reaction of the hybrid protein is carried out at a temperature of 25 ° C with constant stirring. CaCl _{2 was} added to a solution of purified fusion protein to a concentration of 1 mM. Then enterokinase solution (Sigma, USA) was added to the reaction mixture at the rate of 1 U per 4 mg of protein and incubated for 16 hours. The hydrolysis products of the hybrid protein are analyzed by RP-HPLC. The cleavage reaction is stopped by acidifying the material 2 N. hydrochloric acid to a pH of 4.0-5.0.

Information sources

1. RF patent 2203949. A method of obtaining human proinsulin.

2. US patent 5952461. Process for preparing human proinsulin.

3. European patent EP1042479. A process for preparing human proinsulin.

4. RF patent 2263147 C1, 10.27.2005. Bull. Number 30.

5. Twigg A.J. and Sherrat D. // Nature, 1980, v. 283, p. 216-218.

6. Brosius J., Dull T.J., Sleeter D.D., Noller H.F.//J. Mol. Biol., 1981, v. 148, p. 107-127.

7. Laemmli U.K. // Nature, 1970, v. 227, p. 680-687.

Claims (4)

1. The bacterial strain Escherichia coli JM109 / pHINS21 is a producer of a hybrid protein containing human proinsulin. 2. A method of obtaining human proinsulin, including
the cultivation of the producer strain according to claim 1,
destruction of bacterial cells,
isolation of inclusion bodies, their dissolution in a buffer containing urea and dithiothreitol,
renaturation and purification of the renatured hybrid protein with human proinsulin,
cleavage of the hybrid protein by enterokinase or its catalytic subunit,
purification and obtaining the target product. 3. The method according to claim 2, characterized in that
The proinsulin obtained after enzymatic cleavage is purified by ion exchange chromatography on sorbents with sulfopropyl groups. 4. The method according to claim 3, characterized in that
after enzymatic cleavage of the hybrid protein, proinsulin is purified by chromatography on SP-Sepharose FF in 0.05-0.2 M ammonium acetate buffer, pH 3.0-6.0, containing 1-2 M urea, and the sorbed protein is eluted with a linear gradient of potassium chloride from 0 to 0.5 M in a balancing buffer.

Images